1. Field of the Invention
The present invention relates to a method for measuring stable-type hemoglobin in blood and an apparatus for measuring the same.
2. Description of the Prior Art
After blood sugar enters into red blood cells depending on its concentration, the sugar is bound with hemoglobin to form glycohemoglobin. It is indicated that the concentration of stable-type A1c (referred to as stable-A1c or abbreviated as s-A.sub.1c hereinafter) among various types of glycohemoglobin reflects the average blood glucose level for the latest 1 to 3 months.
A.sub.1c is composed of stable-type A.sub.1c (s-A.sub.1c) and labile-type A.sub.1c (labile-A.sub.1c ; l-A.sub.1c). Of the two, s-A.sub.1c exhibits good correlation with the blood glucose level for the latest 1 to 3 months. It is suggested that l-A.sub.1c is about 10 to 15% of the total A.sub.1c in a hungry healthy adult. The l-A.sub.1c is formed by the reversible binding of N terminus of .beta.-chain hemoglobin and a reducing terminus of glucose through a Schiff base, and is generated and decomposed in a relatively short period. In diabetic patients, the l-A.sub.1c content is therefore higher than in healthy adults, sometimes amounting up to 10 to 20% of the total A.sub.1c. It is also higher after meal than in hunger, and is remarkably influenced by the state when blood is collected.
Alternatively, s-A.sub.1c is gradually generated from l-A.sub.1c, continuously and irreversibly. It is suggested that s-A.sub.1c reflects very well the blood glucose level for the latest 1 to 3 months.
Hence, it is preferable that s-A.sub.1c is separated and measured singly. However, the two are extremely analogous in structure, so they are considerably hard to separate by liquid chromatography.
The method of measuring only s-A.sub.1c includes two methods; (1) a method of separating and measuring s-A.sub.1c and l-A.sub.1c on a separation column by chromatography; and (2) a method of pretreating and removing l-A.sub.1c.
In the method for separating l-A.sub.1c and s-A.sub.1c on a separation column, a long, highly preparative separation column is employed for improving preparative performance. The method has a characteristic property in that hemoglobin is hardly denatured compared with the method to remove l-A.sub.1c by its pretreatment (see for example, Gazette of Japanese Patent Laid-open No.75558/1988).
On the other hand, the method to chemically remove l-A.sub.1c by its pretreatment includes the method to incubate red blood cells in physiological saline or a buffer containing semicarbazide and aniline (D. M. Nathan, Clin. Chem., 27, 1261 (1981); D. M. Nathan, et al., Clin. Chem., 28,512 (1982)). Such method focuses on the evidence that the temporary binding of l-A.sub.1c, i.e. the binding through a Schiff base, readily decomposes.
It has been proposed also a method comprising adding a commercially available reagent to blood for removing l-A.sub.1c, and subsequently heating the blood at about 50.degree. C. for 1 to 2 minutes, thereby removing l-A.sub.1c (see for example, Gazette of Japanese Patent Laid-open No. 36143/1988; Gazette of Japanese Patent Laid-open No. 97857/1989).
Of the prior art techniques, however, the complete separation of l-A.sub.1c and s-A.sub.1c according to the method of separating l-A.sub.1c and s-A.sub.1c on a separation column requires an analytical time as long as about 10 to 60 minutes per one sample. Therefore, it is nearly impossible to treat a large number of samples. Because such column is so long that it requires a large amount of fillers, the method has disadvantages that the column costs high and that the apparatus therefor should be bulky.
According to the method of removing l-A.sub.1c by its pretreatment, the pretreatment per se is complex and requires 30 minutes to four hours. The method has a problem also in treating many samples.
Further, the method of heat treatment has a problem that analysis may eventually be impossible in case that the protein in blood is denatured resulting in the occurrence of precipitation, which may clog pipes and filters.